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Multiplex protein detection with the ECL Plex fluorescent Western blotting system using the Ettan DIGE Imager

Key words: ECL Plex • Ettan DIGE Imager • fluorescent Western blotting • multiplex detection • dynamic range

The Ettan™ DIGE Imager is fully compatible with the ECL Plex™ system, reaching the same levels of sensitivity, linearity, and dynamic range as the Typhoon™ scanner. Similar results are also obtained with both scanners in multiplex applications, where two proteins can be detected in the same blot with minimal cross-reactivity between antibodies or dyes.

The ECL Plex Western blotting system is based on the sensitive CyDye™ conjugated antibodies, enabling detection in the low picogram range with high linearity and a dynamic range of nearly four orders of magnitude (1). Multiplexing is another important property of the ECL Plex system, enabling blotting of two proteins simultaneously.

In an ECL Plex experiment, the data quality is not only dependent on the blotting, but also the imaging system used. A new scanning CCD camera, the Ettan DIGE Imager, is now available. It is a camera with high resolution specifically developed for 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) and the ECL Plex system, with the ability to produce multichannel images of Cy™2-, Cy3-, and Cy5 labeled gels and protein blots.

In this study we present a comparison of the Typhoon and the new Ettan DIGE Imager applied to the ECL Plex system. A number of CyDye conjugates were evaluated, using both the Hybond™ ECL (nitrocellulose) and the new low-fluorescent Hybond-LFP (PVDF) membranes in both single-protein and multiplex analyses. The blot scans were evaluated, resulting in data that was comparable between the two scanners, showing sensitive detection and a broad dynamic range. An application of the ECL Plex system with TGF-β–activated cells also resulted in quantitative data for both scanners.

Products used

ECL Plex goat-α-mouse IgG-Cy3, 150 μg PA43009

ECL Plex goat-α-rabbit IgG-Cy5, 150 μg PA45011

ECL Plex goat-α-mouse IgG-Cy5, 150 μg PA45009

Hybond ECL, 20 cm x 3 m RPN203D

Hybond-LFP, 20 x 20 cm, 10 sheets RPN2020LFP

ECL Plex Fluorescent Rainbow™ Markers, RPN851
full range, 500 μl

2D Quant Kit 80-6483-56

miniVE Vertical Electrophoresis system 80-6418-77

EPS 301 Power Supply 18-1130-01

TE 22 Mini Tank Transfer Unit 80-6204-26

Ettan DIGE Imager, including installation kit 63-0056-42

Ettan DIGE Imager Cassette, 11-0027-33
with low-fluorescent glass, for naked gels

Typhoon 9410 9410-PC
(includes ImageQuant™ TL software)

PlusOne™ Bromophenol Blue 17-1329-01

PlusOne DTT 17-1318-01

Plu sOne Glycerol 17-1325-01

PlusOne Glycine 17-1323-01

PlusOne SDS 17-1313-01

PlusOne Tris 17-1321-01

Other materials required
Human apotransferrin (Calbiochem) 616395

Bovine cardiac muscle actin (Sigma-Aldrich) A3653

Rabbit polyclonal anti–human transferrin A0061
(Dako Cytomation)

Monoclonal Anti–Actin, mouse-α-bovine A4700

Monoclonal Anti-β-Tubulin, mouse T4026

Phospho-p38 MAP Kinase 9211S
(Thr180/Tyr182) Antibody,
rabbit (Cell Signaling)

10x PBS (Medicago) 12-9423-5

Novex™ 12% Tris-glycine gel (Invitrogen) EC60055BOX

Methanol (Merck) K33730207

Tween™ 20 (Merck) 8.22184.1000

Methods—single protein detection
Gel electrophoresis

Human apotransferrin was loaded onto Novex 12% Tris-glycine gels in a series of two-fold dilutions from 5 ng to 0.6 pg. Bovine cardiac muscle actin was loaded in a two-fold dilution series from 150 ng to 18 pg. Gel electrophoresis was performed for 2.5 h at 100 V using the miniVE Vertical Electrophoresis System.

Protein blotting and fluorescent detection
After electrophoretic separation the gels were blotted onto either Hybond ECL (low-fluorescent nitrocellulose) or Hybond-LFP (low-fluorescent PVDF) membranes for 2.5 h at 25 V using a TE 22 Mini Tank Transfer Unit followed by incubation in PBS + 0.1% Tween-20 (PBST) blocking solution overnight at 4 °C.

The blots were then incubated with the rabbit anti–human transferrin or the mouse anti–actin primary antibody (1:750 dilution in PBST) for 1.5 h at room temperature. They were washed twice quickly, then twice for 5 min each in PBST, and then incubated for 1 h, protected from light, with the appropriate secondary antibody: ECL Plex goat-α-rabbit IgG-Cy5, ECL Plex goat-α-mouse IgG-Cy5, or ECL Plex goat-α-mouse IgG-Cy3 (1:2500 dilution in PBST).

The membranes were then washed three times quickly, then four times for 5 min each in PBST followed by two brief washes in PBS before scanning on both the Ettan DIGE Imager and the Typhoon scanner. Hybond-LFP membranes were dried at 40 °C for 1 h before scanning, whereas the Hybond ECL membranes were stored in PBS and scanned wet.

On the Ettan DIGE Imager, the Cy3 conjugate was scanned using a 540/25 excitation filter and a 595/25 emission filter, while the Cy5 conjugates were scanned with a 635/30 excitation filter and a 680/30 emission filter. Exposure levels were adjusted until the most intense band reached near saturation. Imaging on the Typhoon scanner was performed using the 633-nm (red) laser with a 670BP30 filter for Cy5-conjugated antibodies and the 532-nm (green) laser with a 580BP30 filter for Cy3 conjugates. The PMT value was adjusted until the most intense band nearly reached saturation. The images were then analyzed using ImageQuant software to determine the limit of detection, linearity, and the dynamic range.

Methods—multiplex protein detection
Model system

A protein mixture of human apotransferrin and bovine cardiac muscle actin was loaded onto Novex 12% Tris-glycine gels in four-fold dilutions from 5 ng to 1.2 pg (transferrin) and in two-fold dilutions from 150 ng to 2.34 ng (actin). The electrophoresis, protein transfer, blocking, and washing steps were identical to the single protein detection protocol. Both the Hybond ECL and Hybond-LFP membranes were used.

After the blocking step, the blots were incubated in a mixture of rabbit anti–human transferrin and mouse anti–bovine actin primary antibodies (diluted 1:750 in PBST) for 1.5 h at room temperature. After washing in PBST, the blots were incubated with a mixture of the secondary antibodies, ECL Plex goat-α-rabbit IgG-Cy5 and ECL Plex goat-α-mouse IgG-Cy3 (both diluted 1:2500 in PBST), on a shaker for 1 h at room temperature and protected from light.

The membranes were then scanned on the Ettan DIGE Imager and the Typhoon scanner in single scans applying both Cy3 and Cy5 wavelengths and filters. Limit of detection and cross-reactivity between the two CyDye-antibody conjugates were analyzed for all alternatives.

TGF-β is a potent growth factor stimulating a number of cellular responses including growth inhibition, cell differentiation, and apoptosis. In this application, human T293 kidney epithelial cells were activated with TGF-β and harvested at different time points. Lysates of these cells wer e prepared, run on gels, transferred to Hybond membranes, and then blotted according to the protocol previously described. The targeted protein was the phosphorylated form of p38 (pp38), a low-abundant protein mediating TGF-β activation. As primary antibodies we used monoclonal anti-actin and phospho-p38 MAP kinase antibody targeted with ECL Plex goat-α-mouse IgG-Cy3 and ECL Plex goat-α-rabbit IgG-Cy5, respectively. The experiments were performed on both Hybond ECL and Hybond-LFP membranes.

Results and discussion
Single protein analysis
All three ECL Plex CyDye conjugates were evaluated on both Hybond ECL and Hybond-LFP membranes scanned on both scanners. The image analysis was performed in the same fashion regardless of scanner used to ensure a fair comparison. A compilation of the results is presented in Table 1. Figure 1 displays selected membrane images with linearity charts below.

Our results demonstrate that the Ettan DIGE Imager generates comparable data to the Typhoon scanner in terms of sensitivity, dynamic range, and linearity. A slightly higher sensitivity was achieved on the Typhoon scanner for some of the membranes, but the lower sensitivity limit of the two scanners can be considered as equal. The differences in dynamic range correspond to the detection limit, while there is no significant difference in linearity between the two scanners. Note the wide dynamic range: up to 3.9 orders of magnitude (8000-fold). The differences in dynamic range between the conjugates reflect innate sensitivity differences. In a single-protein application, if the highest sensitivity level is desired, we recommend ECL Plex goat-α-rabbit IgG-Cy5.

To determine the linearity of the data the square of the correlation coefficient (R2) was used. Pixel intensities fro m the scanned membranes were plotted against the amount of protein loaded onto the gel, and a linear curve fit was performed. The resulting graphs for ECL Plex goat-α-rabbit IgG-Cy5 are displayed below the blot images in Figure 1. A sensitivity of 0.6 pg was reached with both scanners, and the linearity values are in the same range.

Multiplex model system
Using a mixture of ECL Plex goat-α-mouse IgG-Cy3 and ECL Plex goat-α-rabbit IgG-Cy5 fluorescent antibodies, actin and transferrin were simultaneously detected in one blot. Detection was performed on both membrane types (Hybond ECL and Hybond-LFP), and each membrane was scanned on both the Ettan DIGE Imager and Typhoon scanner. Figure 2 displays the overlaid color images from the Cy3 (green) and Cy5 (red) channels. The detection limit was identical for both the Ettan DIGE Imager and the Typhoon scanner, confirming the high performance level of the Ettan DIGE Imager.

Multiplex application
In addition to the multiplex model system, a multiplex application was evaluated on the two scanners. Figure 3 shows the ECL Plex system applied to TGF-β–mediated phosphorylation. A TGF-β–activated cell line was targeted with antibodies against actin and pp38, probing the phosphorylation of p38. Quantitation of activation was also determined, where increasing time of TGF-β stimulation results in an increase of pp38. The comparison between the Ettan DIGE Imager and the Typhoon scanner shows that both scanners deliver quantitative data (Fig 3). These results clearly indicate the quantitation power of the ECL Plex system in combination with the Ettan DIGE Imager.

The high level of performance of the Ettan DIGE Imager has clearly been demonstrated. The Ettan DIGE Imager is fully compatible with the ECL Plex system. The instrument reaches a detection limi t similar or equal to the Typhoon scanner. In a multiplex application, the performance of the Ettan DIGE Imager is fully comparable to that of the Typhoon scanner.

1. Application note: Multiplex protein detection using the ECL Plex fluorescent Western blotting system, GE Healthcare, 28-4015-40, Edition AA (2005).

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